Anti-atherogenic functions of HDL cholesterol include mediation of reverse cholesterol transport as well as anti-oxidant and anti-inflammatory effects. Scavenger receptor class B type-l (SR-BI) and apolipoprotein E (apoE) both play crucial roles in HDL metabolism and reverse cholesterol transport. Combined deficiency of SR-BI and apoE (DKO) in mice results in dyslipidemia, characterized by large free cholesterol (FC)-enriched HDL particles, accelerated occlusive coronary disease, and premature death due to myocardial infarction. Our preliminary studies show that DKO HDL particles may be dysfunctional and proatherogenic by inducing FC overload in wild type macrophages. In Specific Aim (SA) 1, we will examine the hypothesis that the DKO HDL cholesterol particles are severely dysfunctional and proatherogenic, promoting oxidation, inflammation, and abnormal reverse cholesterol transport. ApoE may mediate RCT through both SR-BI dependent and independent pathways, and the loss of both pathways may contribute to the lethal phenotype in DKO mice. Macrophage-specific deletion of either apoE or SR-BI promotes atherosclerosis. Our preliminary studies indicate that macrophage deficiency of both apoE and SR-BI results in dramatically impaired cholesterol homeostasis and a striking increase in accumulation of apoptotic cells in atherosclerotic lesions, independent of the DKO lipid environment. These results suggest that DKO macrophages may be prone to FC-induced apoptosis and/or impaired phagocytic clearance of apoptotic cells in atherosclerosis. The goal of SA2 is to test the hypothesis that macrophage apoE and SR-BI cooperate to limit atherogenesis and apoptosis. The abnormal cholesterol homeostasis of DKO macrophages suggests that SR-BI and apoE function in a cooperative fashion to optimize macrophage trafficking and mobilization of cholesterol. In SA3, in vitro studies will examine the hypothesis that SR-BI-associated cholesterol domains and signaling modulate apoE secretion. Studies will also test the hypothesis that apoE containing HDL are the most efficient acceptors of SR-BI mobilized cholesterol. Finally, we will examine the hypothesis that SR-BI and apoE expression impact ABCA1- and ABCG1-mediated trafficking and mobilization of cholesterol. New insights into the roles of apoE and SR-BI as determinants of the anti-atherogenic functions of HDL cholesterol and macrophage cholesterol homeostasis and apoptosis may lead to new therapeutic approaches for atherosclerosis.